Three predominant isoenzymes of creatine kinase (CK) are recognized; these are dimers consisting of the M and B sub-units. These dimers may comprise two M or two B sub-units, or one M and one B sub-unit. The predominant dimer present in the blood, serum, or plasma of normal individuals is CK-MM isoenzyme, with variable but usually only trace quantities of CK-MB that indicate the normal degradation of skeletal muscle. The CK-BB isoenzyme is not usually present in detectable amounts in serum of normal individuals but is present in significant quantities in brain tissue and smooth muscle.
Elevations of the CK-BB isoenzyme can occur in pathologic conditions such as metastatic carcinoma or severe burns. The presence of elevated levels of CK-MB isoenzyme has been used as a clinically important indication of myocardial infarction in instances where possible sources of significant skeletal muscle damage can be eliminated. More particularly, repetitive determinations of CK-MB levels in serum can indicate the time course and severity of infarctions. Differentiation between the isoenzymes of creatine kinase, therefore, is clinically important and the availability of a rapid, efficient, and highly discriminatory assay for the CK isoenzymes is needed.
Thrombolytic therapy has become standard care in the treatment of acute myocardial infarction (AMI). To be maximally effective, therapy must be implemented in the initial hours of infarction, so the decision to treat cannot be based upon conventional biochemical markers of AMI, such as total CK and/or CK-MB activities, which may remain within the normal range for six hours or more after the onset of symptoms. Consequently, treatment, which carries some risk of hemorrhage, must be implemented on the basis of clinical and electrocardiographic data, which are of limited diagnostic accuracy in the early hours of AMI. Thus, there are continuing efforts to develop techniques that would more quickly provide a reliable diagnosis.
The isoforms of CK have received recent attention as potential markers for early diagnosis of AMI and for assessing the success of thrombolytic therapy. There are three isoforms of CK-MM; CK-MM1, CK-MM2 and CK-MM3; and two isoforms of CK-MB; CK-MB1 and CK-MB2. The isoforms differ in the presence or absence of a C-terminal lysine residue on the M-subunits of the isoforms. CK-MM3 and CK-MB2, the isoforms present in myocardial tissue, have a C-terminal lysine residue on each M-subunit of the isoform. After AMI, CK-MM and CK-MB are released from injured myocardium into plasma. Over time, the myocardial tissue isoforms convert to the plasma isoforms. CK-MM3 first converts to CK-MM2 which, in turn, converts to CK-MM1 and CK-MB2 converts to CK-MB1. The conversion occurs as plasma carboxypeptidase cleaves the C-terminal lysine residues from the M-subunits. Shortly following AMI, the ratio of the tissue isoforms of CK-MM and CK-MB to the plasma isoforms increases. Such increase occurs several hours before either total CK or CK-MB activity exceeds normal reference levels.
Several approaches to analysis of CK isoenzymes and isoforms have been used which rely either upon physical separation of the isoenzymes or isoforms with subsequent identification, or upon highly selective reactions between the isoenzymes or isoforms and antibodies. Physical separation methods such as electrophoresis or column chromatography are time consuming, require considerable skill, and are frequently incapable of highly reproducible separations to resolve adequately the isoenzymes with sufficient sensitivity to monitor early changes in CK-MB levels. The inconvenience of physical separation techniques and their inability to unequivocally resolve CK isoenzymes lead to immunochemical techniques which, based upon their unique structural or immunochemical determinants, have the potential to differentiate between the isoenzymes in complex mixtures.
U.S. Pat. No. 4,260,678, issued Apr. 7, 1981 to Lepp et al., discloses a CK assay using immobilized antibodies to either the CK-M or CK-B sub-unit, or both. The selected antibody, which did not inhibit or substantially change the activity of the bound sub-unit, was immobilized on a carrier such as porous glass beads and then reacted with sample followed by the separation of the immobilized antibody-isoenzyme complexes from the reaction mixture prior to the determination of enzyme activity of sub-units bound to the carrier. This approach is useful to determine total activity by reacting both anti-CK-M and anti-CK-B carriers with sample, or of individual sub-units by reaction of one or the other carrier with sample. It is not possible to determine the activity associated with the hybrid dimer CK-MB or CK isoforms using this approach.
U.S. Pat. No. 4,387,160, issued Jun. 3, 1983 to Gomez et al., discloses an assay for CK-MB which uses three separate antibody preparations and two separate assays on a given sample. In one assay, a precipitating anti-CK-M antibody capable of substantially or completely inhibiting the M-subunit activity, without significantly affecting B-sub-unit activity, is combined with one portion of a sample and allowed to react. If precipitation occurs during this reaction, the precipitate will remain homogeneously suspended during the process of this reaction. The residual isoenzyme activity in this solution is determined by conventional means. A second portion of sample containing at least CK-M sub-units is reacted with an anti-CK-M antibody and the complexes so formed are further reacted with a precipitating second antibody capable of reacting with determinants on the anti-CK-M antibody. The precipitate is separated from the reaction mixture and residual activity, presumably representing B sub-unit remaining in the supernatant, is determined by conventional means. Activity from the second sample representing contaminating B sub-units is subtracted from the activity of the first sample, which represents both MB and contaminating B sub-units, to determine MB activity. This assay combines immunoinhibition with precipitation techniques to determine CK-MB levels in samples, but does so with considerable inconvenience in time-consuming processing steps and with several, highly specialized reagents.
Sandwich immunoassays employing the two-site immunometric approach to measure mass concentrations have been used to detect CK-MB by immobilizing either all M or all B-containing sub-units onto a solid phase through an antibody specific for that sub-unit attached to the solid phase (see U.S. Pat. No. 4,376,110, issued Mar. 8, 1983 to Davis et al., and U.S. Pat. No. 4,624,916, issued Nov. 25, 1986 to Shah et al.). The immobilized sub-unit is then detected by a labelled second monoclonal antibody specific for that sub-unit. The amount of label detected is a direct measure of the desired sub-unit in the original sample. This approach requires two highly specific reagents and is subject to an elevated number of false positive results due to the nonspecific binding of the labelled antibody to the solid phase.
Landt et al., Clin. Chem., 34(3): 575-581 (1981), disclose a colorimetric assay for CK-MB isoenzyme which is a modification of a previously published method [Vaidya et al., Clin. Chem., 32: 657-663 (1986)] based on the use of a monoclonal antibody specific for the CK-MB isoenzyme. Polystyrene beads were coated with the CK-MB-specific monoclonal antibody and then mixed with a serum sample to extract CK-MB. The beads were washed to remove unbound CK isoenzymes and measured for bound CK-MB activity. This method is limited to the determination of CK-MB isoenzyme and cannot be used for the CK isoforms. Furthermore, this technique is not useful in automated analyzers which are limited to the analysis of enzymes in free solution.
U.S. Pat. 4,810,639, issued Mar. 7, 1989 to Pankratz, discloses an immunoassay for CK-MB based on sequential immunoinhibition first by an immobilized and then by a soluble antibody of either the CK-M or CK-B subunits of CK-MM, CK-MB, and CK-BB, followed by an enzymatic determination of the unbound sub-unit in CK-MB; the unbound CK-BB or CK-MM having been previously removed. It is not possible to determine the activity associated with the CK isoforms using this method.
The techniques mentioned thus far are applicable only to the analysis of CK-MB isoenzyme and do not provide for the determination of CK isoforms; thus they have limited utility in the early diagnosis of AMI, when either total CK or CK-MB activity is still within the normal reference range.
Puleo et al., Clin. Chem., 35: 1-4 (1989), disclose an assay based on high voltage gel electrophoresis which can quantitatively determine CK-MB isoforms in plasma at activities as low as 1.25 U/L. This method is of limited utility in a clinical setting due to shortcomings associated with physical separation methods such as lack of reproducibility and the high level of skill required for successful completion.
European Patent application Publication Number 304,628, published on Mar. 1, 1989, discloses an immunoassay for determining the levels of CK-MM.sub.A (CK-MM1), CK-MM.sub.B (CK-MM2), and CK-MM.sub.A and CK-MM.sub.B combined, using antibodies specific for the two isoforms and an antibody selective for both the CK-MM.sub.A and CK-MM.sub.B isoforms but not CK-MB or the CK-MB isoforms. An immunoassay is described which includes immobilization of a selected antibody onto an insoluble solid support, incubation of the antibody-coated support with sample to allow for extraction of the isoform of interest, and, finally, determination of the isoform while bound to the support. Other formats, including sandwich immunoassays and competitive schemes, are also described. This method is not useful for the determination of total CK-MB or the CK isoforms in automated analyzers which are limited to the analysis of enzymes in free solution.
U.S. Pat. No. 4,231,999, issued Nov. 4, 1980 to Carlsson et al., discloses a method for carrying out immunoassays based on the formation of an insoluble conjugate having incorporated into it an analytically indicatable group which is subsequently separated as part of a fragment of the conjugate and assayed in the liquid phase. Fragments containing the analytically indicatable group are formed by splitting the insoluble conjugate at bonds that are covalent in nature, where the splitting of such bonds does not reduce the activity of the analytically determinable group or damage a reagent that comprises a labelled immunochemical component.
Thus, this method requires the use of a labelled reagent. Additionally, since this immunoassay measures the amount of label in solution, the measurement is really one of mass concentration rather than direct activity of the desired analyte. There is some debate as to whether mass measurement is as accurate as CK activity measurement in determining actual CK-MB isoenzyme or CK isoform content.
There is a need for a rapid CK isoenzyme and CK isoform assay that can provide precisely and economically an accurate evaluation of CK isoenzyme or CK isoform levels in body fluids of an individual suspected of suffering from acute myocardial infarction. Further, there is a need for a CK isoenzyme and CK isoform assay that is useful with automated analyzers which are limited to the analyses in free solution.